Bone tissue regeneration is necessary to restore skeletal function following a variety of clinical procedures, including bone fracture repair, reconstructive surgery, and spinal fusions. Tissue-engineered bone offers a number of advantages over traditional bone grafts used for the repair and regeneration of bone. However, the current methods employed to evaluate and optimize the growth of engineered bone tissue lack the spatial and temporal resolution to characterize the dynamic cell-matrix interactions that occur during tissue development. The objective of this project is to develop quantitative optical biomarkers to identify and monitor changes in the biochemical, microstructural, and overall mechanical properties of engineered bone tissue during its in vitro development. This work focuses on the dynamic changes that occur as three-dimensional silk scaffolds seeded with human mesenchymal stem cells develop into functional bone tissue. The central hypothesis of this proposal is that endogenous optical signals can be measured non-invasively using multi-photon imaging and depth-resolved light scattering spectroscopy to determine the biochemical and microstructural properties of the developing tissue. To test this hypothesis, the intrinsic fluorescence and light scattering signals from different cellular and extracellular matrix components will be identified in Aim 1 and correlated with traditional histological and molecular biology techniques. The optical biomarkers for microstructural organization identified in Aim 1 will be used to predict the overall mechanical function of the bone tissue in tension and compression in Aim 2. Collectively, these aims will provide a unique understanding of how the biochemical status and mechanical function of engineered tissue changes during osteogenesis. By using only non-invasive techniques that identify intrinsic sources of optical contrast, the outcomes of this proposed research can be used to optimize the future approaches to engineering functional bone tissue and will enable a means to monitor engineered constructs as they are incorporated into native tissue following surgical repair.